Modulation of gene transcription plays an integral role in human health and, dysregulation of this process has been shown to lead to serious pathological disease states. The majority of current research in the field of transcription is focused on understanding the mechanisms which regulate expression of specific genes. Consequently, global regulatory mechanisms of gene transcription have not received much attention. The research in this proposal will investigate the role that TBP (TATA-box Binding Protein) plays in the global regulation of gene transcription. TBP is the only common subunit linking gene transcription by all three DNA dependent RNA polymerases (RNAP I,II,III). As such, TBP holds a unique, central position in eukaryotic transcription. Data is presented in this proposal which indicates that the amount of TBP in the cell is limiting for transcription, relative to the large number of pathway specific TBP binding proteins termed TAFs (TBP Associated Factors). This result is consistent with a large body of published biochemical and genetic data. Hence, a detailed understanding of the rules which dictate pathway specific TBP-TAF distribution and interaction dynamics could provide insights into gene transcription at a very fundamental level. This grant utilizes a tightly orchestrated combination of genetic and biophysical methods to directly examine TBP-TAF interaction kinetics both in vitro and in vivo. The central hypothesis to be tested is "That the ratio of mRNA and tRNA gene transcription inside the cell is governed by the on-rates, dissociation rates, relative affinities, and Intracellular concentrations of TBP and the cognate TAFs associated with these two pathways." To examine this process the following lines of investigation will be pursued: 1. The intracellular concentration of TBP and the TAFs in yeast cells will be accurately determined. 2. Region-directed mutagenesis methods will be used to generate TBP- binding mutants of the genes encoding a rationally selected subset of yeast RNA Pol II and Pol III TAFs. 3. Stopped-flow fluorescence anisotropy assays will be performed to examine the assembly/disassembly kinetics of each TBP-TAF complex. 4. Yeast cells expressing mutant TAFs will be subjected to a combination of molecular genetic and in vivo fluorescence measurements to demonstrate that changes in TBP-TAF interaction kinetics measured in vitro, result in the predicted change in both TBP-TAF distribution pattern and gene transcription levels in vivo.